Study on the in vitro activity of Hehuan Yin aqueous extract against hepatitis C 2a virus

Yuan-Yuan Wu,Yong-Lin Liang,Fei-Long Chen,Qing-Fa Tang

School of Traditional Chinese Medicine,Southern Medical University,Guangdong Provincial Key Laboratory of Chinese Medicine Pharmaceutics,Guangdong Provincial Engineering Laboratory of Chinese Medicine Preparation Technology,Guangzhou 510515,China

Keywords:Hehuan Yin aqueous extract Hepatitis C 2a virus In vitro experiments Antiviral activity

ABSTRACT Objective:To study the anti-HCV activity and mechanism of Hehuan Yin aqueous extract.Methods:Huh7.5.1 cells were used to establish the HCV2a virus infection model.Cell survival rate (%) and Renilla Luciferase Assay Kit (%) were calculated by Celltiter-GLO Assay for evaluating CC50,EC50 and SI values.To observe the drug resistance of the virus to different concentrations of Hehuan Yin within 72 hours by detecting luciferase activity,western-blot was used to detect the protein expression levels of NS5A,NS3 and NS5B.Results:the CC50,EC50 and SI of Hehuan Yin against HCV2a were 132.50g/ml,1.90g/ml and 67.90 respectively.The EC50 after 24h,48h and 72h administration were 18g/ml,5.8g/ml and 2.3g/ml respectively.Within the range of drug concentration,the aqueous extract Hehuan Yin had inhibitory effect on the expression of NS5A and NS5B proteins in a doseeffect relationship,but had no obvious effect on the expression of NS3 protein.Conclusion:The aqueous extract of Hehuan Yin may inhibit the replication of HCV2a virus by changing the protein expression levels of NS5A and NS5B,and the virus has no tolerance to the aqueous extract of Hehuan Yin.

1.Introduction

Hepatitis C virus (HCV) belongs to the Faviviridae family.Its genome is a single stranded RNA[1] containing a 5' coding region,an open reading frame and a 3' coding region.After encoding in the open reading frame,it is hydrolyzed into 3 structural proteins (core protein,glycoprotein,E1 and E2) and 7 non-structural proteins(NS1,NS2,NS3,NS4A,NS4B,NS5A and NS5B)[2].There are 6 genotypes of hepatitis C

Virus,mainly 1B and 2A[3] in China.Hepatitis C virus infection can cause hepatitis C.Meanwhile,55%-85% of HCV infected persons can develop into chronic hepatitis or even cirrhosis or liver cancer[4,5],which is very harmful to the health and life of patients.Hepatitis C virus infection mainly involves blood transmission,sexual contact transmission and mother-to-child vertical transmission [6].Clinical anti-HCV drugs are direct anti-HCV drugs(DAAs),which are mainly divided into three categories :NS3/4A serine protease inhibitors,NS5B polymerase inhibitors and NS5A protein inhibitors[7,8].Although it has achieved a good clinical effect,can not restore the liver damage caused by hepatitis C.At the same time,the combination of drugs will bring serious side effects and easy to produce drug resistance.However,traditional Chinese medicine has the advantages of safety and low toxicity and side effects,which has become a new research field to find and develop drugs for HCV treatment.

The HeHuan Yin is composed ofAlbizia JulibrissinandAmpelopsis japonicaaccording to the ratio of 1:1.The use of hehuan Yin has been recorded in the traditional Chinese medicine classics "Shengji Zong Lu" and "Jingyue Quanshu".Albizia Julibrissintaste sweet,flat.It belongs to heart,lung,liver,with soothing liver depression,carbuncle swelling effect.The Ampelopsis japonica taste bitter,simba.It belongs to heart,liver,spleen,with heat detoxification,scattered pain,muscle sore effect.Both theAlbizia Julibrissinand theAlbizia Julibrissinhave anti-tumor [9,10,11],antibacterial[12,13],anti-inflammatory[14],immune regulation [15,16],protection of liver injury [17,18] and anti-oxidation [19,20] and so on.At present,there has been no report on the anti-HCV activity of Hehuan Yinin vitro.Since HCV is only infected in humans and gorillas,and the current animal model is not very mature,this study preliminarily studied the anti-HCV activity of Hehuan Yinin vitroby cell experiment.It is of great clinical significance to provide scientific basis for the search and development of new anti-HCV Chinese herbal compound preparations with low therapeutic cost and effective treatment for HCV patients.

2.Materials

2.1 Cells and virus strains

Hepatocellular carcinoma cell strain HUH7.5.1 and infectious HCV type 2A virus strain were provided by and stored in Wuhan Institute of Virology,CAS.

2.2 Drugs and Reagents

TheAlbizia Julibrissin(batch number:2019-12-07) and theAmpelopsis japonica(batch number:2018-11-09) were both purchased from Anhui Xiehe Cheng Pharmaceutical Company;2' -C-methyl cytidine (2'-CMC) was purchased from Shanghai Yuanye Biotechnology Company;Mouse anti (article No.:D60023)was purchased from Guangzhou Dewei Biotechnology Company.Rabbit Kang (article No.:K106389P) was purchased from Beijing Solaibao Technology Company.Hepatitis C Virus NS5A protein antibody (GTX103358-S),Hepatitis C Virus NS5B protein antibody (GTX103358-S),Hepatitis C Virus NS5B protein antibody(GTX103358-S)GTX131273-S),Hepatitis C Virus NS3 protein antibody (Item No.:GTX131276-S),Genetex,USA.Fetal bovine serum was purchased from Shanghai Yuanye Biological Company.Penicillin,streptomycin and DMEM culture medium were purchased from Gibco.Celltiter-GloTM kit purchased from Promega Company.Renilla Luciferase Assay System kit was purchased from Promega Company.

2.3 Instruments

Model 3111 CO2 incubator,Thermo Fisher Scientific,USA;CKX31 inverted biological microscope,Olympus Corporation,Japan;Synergy 2 multifunctional enzyme plate instrument,Berten Instruments Inc.,USA;N-3000 rotary evaporator,Eyela Corporation,Japan;DZX-6090B Vacuum Dryer,Shanghai Fuma Experimental Equipment Company.BSA224S-CW 1/10000 scale,Germany Sartorius Company;Bole miniature vertical electrophoresis tank,Bole transfer mode electrophoresis tank,Bole Life Medical Products Company;HC-3018R high speed refrigerated centrifuge,Anhui Zhongke Zhongjia Scientific Instrument Company.

3.Methods

3.1 Preparation of HeHuan Yin and 2' -C-methylcytidine

The bark ofAlbizia julibrissinDurazz.was identified by Professor Chao Zhi from Southern Medical University as the dried bark ofAlbizia julibrissin Durazz.Ampelopsis japonica (Thunb.)Makinois the dried root ofAmpelopsis japonica (Thunb.)Makino,a climbing vine of Grapeaceae.TheAlbizia julibrissinand theAmpelopsis japonicawere both purchased from Bozhou Herb Market,Anhui Province.HeHuanYin comprises two herbs:Albizia julibrissin DurazzandAmpelopsis japonica.The raw materials were blended at a ratio of 1:1(w/w).100g herbal mixtures were boiled at 100 temperature for 90 min with distilled water.The extracted aqueous solution was filtered using filtered paper and then evaported with water bath.After vacuum drying for 48h,the final quantity of extracted HeHuan Yin powder was 14.95g.Different concentrations of 2' -C-methylcytidine were prepared with DMEM medium containing 1%DMSO.

3.2 Cells and Treatments

The cell line used for the detection was hepatocellular carcinoma cell line HUH7.5.1,which was susceptible to HCV.The HCV2Ainfected Huh7.5.1 cell model was cultured by adding disease venom into HUH7.5.1 cell line.Huh7.5.1 Cell culture conditions after passage :cultured in DMEM supplemented with 10 % fetal bovine serum,100 U/ ml penicillin and 100 g/ ml streptomycin at 37℃and 5 % CO2 incubator.

3.3 Cytotoxicity of HeHuan Yin

Huh7.5.1 cells were seeded into a 96-well cell culture plate and glued to the wall.The drug was diluted in DMEM medium for 8 gradients at a continuous 3-fold gradient starting from 4 times the highest test concentration.Drugs and appropriate medium were added into the cells and cultured in a CO2 incubator at 37℃.After 72h of culture,the drug-induced cytopathic effect (CPE)was observed under a microscope.After adding celltier-Glo,the absorbance value was measured at 450nm.The toxicity of a drug to a cell is expressed by the activity of the cell.

Calculation formula:cell activity (%)=absorbance value of drug group/average absorbance value of cell control group ×100%.

3.4 Antiviral Activity of HeHuan Yin

Huh7.5.1 cells were inoculated in 96-well cell culture plates overnight for reserve use.The drug dilution ratio and cell culture conditions were the same as 2.3.After 72 h of culture with drug and virus,an appropriate amount of cell solution was added to lyse for 10 min,and then the sea kidney luciferase substrate was added.The Rluc reading value was detected by a multifunctional microplate reader.The experiment set 2 ' -C-methylcytidine as positive drug control,no virus and compound cell hole as cell control,only virus hole as virus control.

Calculation formula:inhibition rate (%)=100-(RLuc value of sample well -RLuc value of cell control)/(RLuc value of virus control -RLuc value of cell control) ×100%

3.5 Virus Resistance

Huh7.5.1 cells were inoculated in 96-well cell culture plates overnight for reserve use.The drug was diluted in DMEM medium for 5 successive gradients,starting from the highest test concentration of 37g/ ml.The cell culture conditions were the same as those in 2.3.After adding the drug 24h,48h and 72h,appropriate amount of cell fluid was added,and after cracking for 10min,luciferase substrate was added.The luciferase activity value was determined by using a multifunctional enzyme plate analyzer.

3.6 Western-blot analysis

The dilution concentration of the drug was set to be the same as 2.5,and the administration method was as follows:2.3.After 72h of culture,the protein was extracted,the sample was prepared for electrophoresis,and the protein was transferred to the PVDF membrane by membrane transfer.5% skim milk was prepared with TBST and sealed for 1 h.After washing the film,corresponding primary antibodies were added (except GAPDH diluted at 1:5 000,other primary antibodies diluted at 1:1 000) and incubated overnight at 4℃.The corresponding rabbit secondary antibody was added for 1h.Film washing,ELC fluorescence color and exposure.

3.7 Statistical analysis

SPSS 23.0 software was used to analyze the experimental data.Cell survival rate,cell inhibition rate and luciferase activity were expressed as mean ± standard deviation ().

4.Results

4.1 Cytotoxicity

The cytotoxicity results showed that the CC50 of the aqueous extract of Huh7.5.1 cells was 132.50g/ ml.The aqueous extract of HeHuan Yin had no cytotoxicity in the concentration range of less than 12.35 g/ mL,indicating 100% cell viability,as shown in Figure 1.

Table 1 Data of Huh7.5.1 Cell Viability of Hehuanyin(n=3,)

Table 1 Data of Huh7.5.1 Cell Viability of Hehuanyin(n=3,)

Figure 1 Curve of Huh7.5.1 cells Viability of Hehuanyin

4.2 Anti-HCV2A virus activity

The experimental results showed that the EC50 of the aqueous extract of Huh7.5.1 cells was 1.95g/ ml,and the EC50 of the positive drug 2' -C-methylcytidine was 0.88g/ ml.In conclusion,the anti-HCV2A activity of HeHuan Yin was similar to that of the positive drug 2' -C-methylcytidine.The anti-HCV2A effect of HeHuan Yin was dose-dependent with its concentration.The experimental results were shown in Figure 2.

Figure 2 Image of inhibition rate of Huh7.5.1 cells infected with HCV2a virus

Table 2 Inhibition rate of Huh7.5.1 cells infected with HCV2a virus (n=3,)

Table 2 Inhibition rate of Huh7.5.1 cells infected with HCV2a virus (n=3,)

4.3 The treatment index TI value of the sample

The therapeutic index (TI) is the safety index of the drug.The ratio of half lethal dose (CC50) to half effective dose (EC50) is often referred to as therapeutic index.WhenTI>2,it is effective.When TI=1,it is low efficiency and high toxicity.WhenTI<1,it is invalid.The therapeutic index TI value of HeHuan Yin was 67.90.The TI value of 2' -C-methylcytidine was 34.00,which was higher than that of the positive drug.BothTI>2.Both HeHuan Yin and the positive drug 2' -C-methylcytidine are effective in inhibiting the replication of HCV virus.

Table 3 Data of CC50、EC50 and TI of samples

4.4 Drug resistance

Drug resistance refers to the resistance of microorganisms,parasites and tumor cells to the action of chemotherapy drugs.Once drug resistance is developed,the chemotherapy effect of the drug is significantly reduced.The half effective concentration of the aqueous extract of HeHuan Yin was 18g/ ml after 24h administration,5.8g/ml after 48h administration,and 2.3g/ ml after 72h administration.Compared with the 50% effective concentration at 24h,the 50%effective concentration at 48h decreased significantly.Compared with the 50% effective concentration at 48h,the 50% effective concentration at 72h decreased significantly.With the prolongation of administration time,the half effective concentration of the aqueous extract of HeHuan Yin decreased gradually.Meanwhile,the anti-HCV effect of HeHuan Yin was increased gradually.This is shown in Figure 3.

Table 4 Effects of different concentrations of HeHuan Yin on luciferase activity within 72h

Figure 3 Time-dose relationship of Hehuan Yin

4.5 Effect of HeHuan Yin on the expression of HCV2A virus related proteins

The aqueous extract of HeHuan Yin was continuously diluted with the highest test concentration of 37.0g/ ml for 5 gradients.Different concentrations of HeHuan Yin had inhibitory effects on the expression of NS5A and NS5B proteins.Within the concentration range,the protein expressions of NS5A and NS5B showed a doseeffect relationship with the concentration of HeHuan Yin.Within the range of diluted concentration,the effect of HeHuan Yin on the expression of NS3 protein was not obvious.There was no significant dose-effect relationship between the expression of NS3 protein and the concentration of HeHuan Yin.This is shown in Figure 4.

Figure 4 Influence of different concentrations of Hehuan Yin on the protein expression levels of NS5A,NS5B and NS3

5.Discussion

Currently,there is no effective vaccine to prevent HCV infection.The main clinical treatment for hepatitis C is direct antiviral drugs(DAAs),which focus on HCV NS3/4A protein (protease),NS5b protein (polymerase) and NS5a protein [21].Some studies have reported that the combination therapy of anti-HCV drugs may lead to an increase in the incidence of early liver cancer [22].Therefore,it is still of great clinical significance to search for and develop new anti-HCV drugs.Traditional Chinese medicine has obvious curative effect on the symptoms of liver disease.Among them,theAlbizia julibrissinand theAmpelopsis japonicabelong to the liver meridian,and HeHuan Yin is traditionally used for the treatment of lung carbuncer.Our study first found that HeHuan Yin has the activity of anti-HCV2A virus.The results of cytotoxicity test showed that there was no cytotoxicity in a certain range.Study showed that HeHuan Yin had anti-HCV2A activity,which was similar to 2' -C-methylcytidine.Therapeutic index is an evaluation index of comprehensive cytotoxicity and antiviral activity [23].TI value indicates that HeHuan Yin is saver than 2' -C-methylcytidine,which has a great advantage in clinical drug safety.At present,the low resistance barrier of DAAS and the existence of mutant strains of RAVS directly lead to the drug resistance problem of DAAS becoming more and more prominent.In the drug resistance experiment,the EC50 after 24h,48h and 72h administration were 18g/ml,5.8g/ml and 2.3g/ml,respectively.EC50 indicated that the virus had no resistance to HeHuan Yin within 72h.The expression of NS5A and NS5B protein was inhibited by aqueous extracts of different concentrations of HeHuan Yin.The study suggests that Huaxia can inhibit HCV virus replicationin vitroand avoid drug resistance of DAAS,which provides a scientific basis for clinical treatment of hepatitis C.

The positive drug used in this study was 2' -C-methylcytidine,a nucleoside compound with anti-HCV activity and a nucleoside inhibitor of HCV NS5B polymerase,which prevents viral production of HCV RNA and thus prevents viral replication [24,25].HeHuan Yin inhibits the expression of NS5A and NS5B proteins and thus achieves antiviral effect by blocking the replication of HCV2A virus in normal cells,which is similar to the antiviral effect achieved by positive drugs that prevent the replication of the virus.The aqueous extract of HeHuan Yin may block the replication of HCV2A virus by inhibiting the protein expression levels of NS5A and NS5B.It has the advantage of no drug resistance to the virus.This provides a scientific basis for the anti-HCV Chinese medicine compound preparation and has important clinical significance.